Aim: To review the accumulated 30 patients with different area of Y chromosome microdeletions, focusing on their correlation with the clinical and pathological findings. Methods: A total of 334 consecutive infertile m...Aim: To review the accumulated 30 patients with different area of Y chromosome microdeletions, focusing on their correlation with the clinical and pathological findings. Methods: A total of 334 consecutive infertile men with azoospermia (218 patients) and severe oligoasthenospermia (116 patients) were screened. Complete physical and endocrinological examinations, general chromosome study and multiplex polymerase chain reaction assay to evaluate the Y chromosome microdeletion were performed. Ten patients received testicular biopsy. Then the clinical and pathological findings were analyzed with reference to the areas of Y chromosome microdeletion. Results: There is a decline of the percentage of sperm appearing in semen in the group that the gene deletion region from AZFc to AZFb. The clinical evidence of the impairment (decreased testicular size and elevated serum FSH) is also relevantly aggravated in this group. However, the pathology of testicular biopsy specimen was poorly correlated with the different deletion areas of the Y chromosome, which may be due to the limited number of specimens. Conclusion: The clinical correlation of spermatogenic impairment to the different AZF deletion regions may provide the information for the infertile couples in pre-treatment counseling.展开更多
Background:Oligoasthenospermia is one of the main causes of male infertility.Researchers usually use chemical drugs to directly damage germ cells to prepare oligoasthenospermia models,which disregards the adhesion and...Background:Oligoasthenospermia is one of the main causes of male infertility.Researchers usually use chemical drugs to directly damage germ cells to prepare oligoasthenospermia models,which disregards the adhesion and migration between spermatogenic cells and Sertoli cells.TAp73 is a critical regulator of the adhesin of germ cell;thus,we sought to explore a novel oligoasthenospermia model based on TAp73 gene suppression.Methods:Mice in the Pifithrin-αgroup were injected intraperitoneally with 2.5 mg/kg Pifithrin-α(TAp73 inhibitor)daily for 30 consecutive days.Reproductive hormone levels and epididymal sperm quality,as well as the network morphology of Sertoli cells were tested.Results:Sperm density,motility,and the relative protein and mRNA expression of TAp73 and Nectin 2 were obviously decreased in the Pifithrin-αgroup compared with the normal control group.No significant distinction was observed in the relative mRNA and protein expression of ZO-1.Furthermore,the tight junctions(TJs)and api-cal ectoplasmic specialization(ES)were destroyed in the Pifithrin-αgroup.Conclusion:The above results indicate that we successfully established a new oli-goasthenospermia mouse model.This study provides a foundation for further explo-ration of the roles of TAp73 genes during spermatogenesis and provides new research objects for further oligospermia research and future drug discovery.展开更多
Objective To evaluate the effect of the accessory gential gland on the male fertility. Methods The relevant seminal parameters of 32 normal donors and 35 patients with olioasthenospermia were measured by seminal routi...Objective To evaluate the effect of the accessory gential gland on the male fertility. Methods The relevant seminal parameters of 32 normal donors and 35 patients with olioasthenospermia were measured by seminal routine analysis, sperm morphology and accessory, gentital glands markers.Results The percentage of normal spermatozoa, the function of sperm penetration through cervical mucus and the enzyme activity of neutral α-1,4-glucosidase significantly declined as compared with the control. There was a significant negative relationship between the semen volume and the sperm motility for the oligoasthenospermia （r= -0.415, P〈0.05）, and a significant positive relationship between the sperm count and the percentage of normal sperm （r=0.393, P〈0.05）.Conclusion The abnormal function of epididymis exists in patients with oligoasthenospermia. Sperm function,for the oligoasthenospermia declines with an increase of the percentage of the abnormal sperm. The poorer the spermatogenises function of the testis is, the higher the percentage of the abnormal sperm appears.展开更多
Objective:To investigate the effect of Xuduan Zhongzi prescription on the expression of HSP60 and HSP90 in testicular spermatogenic cells and semen quality of oligozoospermia and asthenospermia model rats.Methods:A to...Objective:To investigate the effect of Xuduan Zhongzi prescription on the expression of HSP60 and HSP90 in testicular spermatogenic cells and semen quality of oligozoospermia and asthenospermia model rats.Methods:A total of 40 SPF male rats were randomly divided into 10 blank control group and 30 model group.After successful modeling,the model group was divided into model control group,levocarnitine group and Xuduan Zhongzi prescription group with 10 rats in each group.The rats in levocarnitine group and Xuduan Zhongzi prescription group were given corresponding drugs by gavage according to the equivalent measurement of humanbody,the blank control group and model control group were given the same amount of normal saline by gavage,and the rats in each group were killed after 8 weeks of continuous administration.Take the left epididymis of each group for sperm quality detection,take the left testis of each group,observe the histomorphological changes by HE staining,and detect the expression of HSP60 and HSP90 in testicular tissue by immunohistochemistry and Western blot.Results:HE staining showed that compared with the blank control group,the convoluted tubules in the model group were significantly atrophic,degenerated and irregularly arranged,the spermatogenic epithelium showed a large number of vacuoles,Sertoli cell degeneration,obvious inflammatory infiltration in stromal cells,stromal cell proliferation,scattered rows of epithelial cells,a large number of exfoliation and degeneration,and sperm were rare.After the intervention of Xuduan Zhongzi prescription,the above lesions were significantly improved,the structure of seminiferous tubules was relatively regular and orderly,denser than that of the model group,sertoli cells and stromal cells increased significantly,the seminiferous epithelium was thicker than that of the model group,with rich layers,and a large number of sperm could be seen in the lumen.Immunohistochemistry and Western Blot showed that the expression of HSP60 and HSP90 increased significantly after the intervention of Xuduan Zhongzi prescription in model rats(P<0.01).The sperm quality test results showed that compared with the blank control group,the sperm concentration and activity rate in the model group decreased significantly,(P<0.01).After continuous seed cutting,the sperm concentration and activity rate of model rats were significantly increased,(P<0.01).Conclusion:Xuduan Zhongzi prescription may inhibit the apoptosis of testicular spermatogenic cells in oligoasthenospermia model rats by increasing the expression of HSP60 and HSP90,so as to improve sperm quality.展开更多
Objective:To investigate the therapeutic effects and pharmacological mechanisms of Yishen Xingyang capsule(YXC)in oligoasthenospermia(OA)rats.Methods:Forty-eight male SpragueeDawley rats were randomly divided into eig...Objective:To investigate the therapeutic effects and pharmacological mechanisms of Yishen Xingyang capsule(YXC)in oligoasthenospermia(OA)rats.Methods:Forty-eight male SpragueeDawley rats were randomly divided into eight groups of six rats each:normal control(NC);model control(MC);three different positive drug(PD);and low-,medium-,and high-dose YXC groups.A rat model of OA was established by administering glucosides of Tripterygium wilfordii Hook.F(GTW).After YXC administration,penile erectile function was observed.The epididymis,blood,and testes of the rats were harvested for analysis of sperm quality,sex hormone levels,mitochondrial membrane potential,and the transforming growth factor(TGF)-b1/Smad signaling pathway.Results:Compared with that in the MC group,penile erectile function in the YXC groups and three PD groups increased(all P<.01).Moreover,sperm quality in the YXC groups and three PD groups improved(all P<.001).The levels of testosterone,follicle stimulating hormone,and luteinizing hormone in the three PD and YXC groups increased(all P<.05).The mitochondrial membrane potential in the three PD and YXC groups significantly improved(all P<.001).Furthermore,the YXC and three PD groups showed decreased TGF-b1 expression(all P<.05)compared with the MC group.The high-dose YXC group and three PD groups improved Smad2 and Smad4 expression(all P<.05).Conclusion:YXC improved penile erectile function and sperm quality in OA rats,and the underlying mechanism included increase in sex hormones,inhibition of sperm apoptosis,and regulation of the TGFb1/Smad signaling pathway.Meanwhile,this study provides a new effective drug option for the treatment of OA,which is beneficial to male reproductive health and social harmony.展开更多
Objective:To investigate the protective mechanism of Xuduan Zhongzi prescription against epididymal oxidative damage in oligoasthenospermia model rats.Methods:Forty SD rats were randomly divided into blank group,model...Objective:To investigate the protective mechanism of Xuduan Zhongzi prescription against epididymal oxidative damage in oligoasthenospermia model rats.Methods:Forty SD rats were randomly divided into blank group,model group,Xuduan Zhongzi prescription group(10g/kg)and L-carnitine group(0.1g/kg).Except blank group,all induced oligoasmospermia.The blank group and model group were given normal saline intragastric administration,the Xuduan Zhongzi prescription group was given Xuduan Zhongzi prescription solution intragastric administration,and the L-carnitine group was given L-carnitine intragastric administration.HE staining was used to observe the epididymis structure after 8 weeks.The concentration and activity rate of epididymis sperm were measured by sperm quality.MRNA and protein expression levels of Nrf2,NQO1 andγ-GCs in epididymis were detected by RT-qPCR and immunohistochemistry.Results:①HE staining:in the blank group,the epididymis tubes were arranged tightly and regularly,the tissue structure was complete,the epithelial cells were arranged orderly,and the lumen sperm were numerous and evenly distributed.The epididymis of model group showed structural atrophy,loose arrangement,enlarged mesenchyme,increased cell debris and significantly reduced sperm cells.Compared with the model group,the lumen lesions of epididymis in Xuduan Zhongzi prescription group and L-carnitine group were significantly improved,and the amount of normal sperm in lumen was increased and the distribution was uniform.②Results of sperm quality comparison among each group:sperm density and sperm motility rate:compared with blank group,sperm density and sperm motility rate in other groups were significantly decreased(P<0.05),and sperm density and sperm motility rate in model group were significantly decreased(P<0.05);Compared with model group,the sperm density and motility rate in Xuduan Zhongzi prescription group and L-carnitine group were significantly increased(P<0.05).③RT-qPCR and immunohistochemistry:Compared with the blank group,the mRNA and protein levels of Nrf2,NQO1 andγ-GCs in epididymal rats in model group were significantly decreased(P<0.05),while the mRNA and protein levels of Nrf2,NQO1 andγ-GCs were significantly increased in L-carnitine group and Continua seed formula group(P<0.05).Conclusion:Xuduan Zhongzi prescription can reduce oxidative stress damage and improve sperm quality of oligoasthenospermia.The mechanism may related to promoting the activation of Nrf2-NQO1/γ-GCS pathway in epididymis of oligoasthenospermia rats,and up-regulate the expressions of Nrf2,NQO1 andγ-GCS proteins.展开更多
目的基于AMPK介导的线粒体自噬方面探究国医大师王琦院士研发的治疗男性不育的国家中药三类新药黄精赞育胶囊治疗少弱精子症细胞层面的分子机制。方法选择丙烯醛(acrolein,ACR)作用于GC-2spd(ts)小鼠精母细胞制作少弱精子症细胞模型,通...目的基于AMPK介导的线粒体自噬方面探究国医大师王琦院士研发的治疗男性不育的国家中药三类新药黄精赞育胶囊治疗少弱精子症细胞层面的分子机制。方法选择丙烯醛(acrolein,ACR)作用于GC-2spd(ts)小鼠精母细胞制作少弱精子症细胞模型,通过CCK8细胞活性检测确定后续造模用ACR浓度和造模时间。造模成功后加入不同浓度含黄精赞育胶囊的完全培养基进行细胞培养,于24 h后CCK8细胞活性检测,根据细胞活力确定后续给药浓度。GC-2spd细胞贴壁后分为NC组、造模组和ACR+HJZY给药组,通过共聚焦荧光显微镜观测黄精赞育胶囊对线粒体自噬的影响;将上述3组细胞分别转染siRNA-NC和siRNA-AMPK,分为siRNA-NC+control、siRNA-NC+ACR、siRNANC+ACR+HJZY、siRNA-AMPK+control、siRNA-AMPK+ACR、siRNA-AMPK+ACR+HJZY 6组,通过Western blot验证黄精赞育胶囊对AMPK介导的p-AMPK、LC3B、P62、PINK1、Parkin、TBK1、ULK1等线粒体自噬相关蛋白的调控作用。结果通过细胞活性检测实验确定ACR的造模浓度为34μmol/L,造模时间为20 min,HJZY给药浓度为160μmol/L。共聚焦荧光显微镜显示黄精赞育胶囊对受损生精细胞线粒体膜电位具有一定正向调节作用,造模组线粒体膜电位较NC组大幅降低,ACR+HJZY给药组给药后细胞膜电位较造模组有所升高,差异有统计学意义(P<0.05);Western blot检测结果显示siRNA-NC+ACR组p-AMPK/AMPK和PINK1蛋白表达水平较siRNA-NC+control组下降(P<0.001),siRNA-NC+ACR组Parkin蛋白水平较siRNA-NC+control组有所下降,但差异无统计学意义,HJZY给药后这3种蛋白表达水平均较siRNANC+ACR组上升(P<0.001);siRNA-NC+ACR组LC3B、P62、TBK1和ULK1蛋白表达水平均较siRNA-NC+control组上升(P<0.01),siRNA-NC+ACR+HJZY组上述蛋白表达水平均较siRNA-NC+ACR组有所下降(P<0.05)。转染敲减基因siRNAAMPK后,siRNA-AMPK+ACR组p-AMPK/AMPK、PINK1和Parkin蛋白表达水平较siRNA-AMPK+control组有所下降(P<0.01);H J Z Y给药后上述3种蛋白表达水平与s i R N A-A M P K+A C R组比较差异均无统计学意义;s i R N A-AMPK+ACR+HJZY组与siRNA-AMPK+ACR组相比,LC3B蛋白表达水平仍降低(P<0.01),但P62、TBK1和ULK1蛋白表达水平差异均无统计学意义;siRNA-AMPK+control组与siRNA-NC+control组相比,p-AMPK/AMPK、ULK1和TBK1蛋白表达水平下降(P<0.001),PINK1蛋白表达水平亦下降(P<0.05),P62蛋白表达水平上升(P<0.001);siRNA-AMPK+ACR组与siRNA-NC+ACR组相比,TBK1蛋白表达水平下降(P<0.001),LC3B蛋白表达水平下降(P<0.01),ULK1蛋白表达水平亦下降(P<0.05),PINK1和Parkin蛋白表达水平有所下降但差异无统计学意义;siRNA-AMPK+ACR+HJZY组与siRNANC+ACR+HJZY组相比,p-AMPK/AMPK、PINK1和Parkin蛋白表达水平下降(P<0.05),LC3B蛋白表达水平亦下降(P<0.01),P62蛋白表达水平上升(P<0.001),TBK1和ULK1蛋白表达水平差异无统计学意义。结论黄精赞育胶囊可能通过调节AMPK介导的线粒体自噬达到治疗男性少弱精子症的效果。展开更多
Oligoasthenospermia is the primary cause of infertility.However,there are still enormous challenges in the screening of critical candidates and targets of oligoasthenospermia owing to its complex mechanism.In this stu...Oligoasthenospermia is the primary cause of infertility.However,there are still enormous challenges in the screening of critical candidates and targets of oligoasthenospermia owing to its complex mechanism.In this study,stem cell factor(SCF),c-kit,and transient receptor potential vanilloid 1(TRPV1)biosensors were successfully established and applied to studying apoptosis and autophagy mechanisms.Interestingly,the detection limit reached 2.787×10^(-15)g/L,and the quantitative limit reached 1.0×10^(-13)g/L.Furthermore,biosensors were used to investigate the interplay between autophagy and apoptosis.Schisandrin A is an excellent candidate to form a system with c-kit similar to SCF/c-kit with a detection constant(K_(D))of 5.701×10^(-11)mol/L,whereas it had no affinity for SCF.In addition,it also inhibited autophagy in oligoasthenospermia through antagonizing TRPV1 with a K_(D) of up to 4.181×10^(-10)mol/L.In addition,in vivo and in vitro experiments were highly consistent with the biosensor.In summary,high-potency schisandrin A and two potential targets were identified,through which schisandrin A could reverse the apoptosis caused by excessive autophagy during oligoasthenospermia.Our study provides promising insights into the discovery of effective compounds and potential targets via a well-established in vitro-in vivo strategy.展开更多
文摘Aim: To review the accumulated 30 patients with different area of Y chromosome microdeletions, focusing on their correlation with the clinical and pathological findings. Methods: A total of 334 consecutive infertile men with azoospermia (218 patients) and severe oligoasthenospermia (116 patients) were screened. Complete physical and endocrinological examinations, general chromosome study and multiplex polymerase chain reaction assay to evaluate the Y chromosome microdeletion were performed. Ten patients received testicular biopsy. Then the clinical and pathological findings were analyzed with reference to the areas of Y chromosome microdeletion. Results: There is a decline of the percentage of sperm appearing in semen in the group that the gene deletion region from AZFc to AZFb. The clinical evidence of the impairment (decreased testicular size and elevated serum FSH) is also relevantly aggravated in this group. However, the pathology of testicular biopsy specimen was poorly correlated with the different deletion areas of the Y chromosome, which may be due to the limited number of specimens. Conclusion: The clinical correlation of spermatogenic impairment to the different AZF deletion regions may provide the information for the infertile couples in pre-treatment counseling.
基金This project was supported by the Natural Science Foundation of Anhui Provincial Department of Education(no.KJ2020A0386)National Natural Science Foundation of China(no.82174162).
文摘Background:Oligoasthenospermia is one of the main causes of male infertility.Researchers usually use chemical drugs to directly damage germ cells to prepare oligoasthenospermia models,which disregards the adhesion and migration between spermatogenic cells and Sertoli cells.TAp73 is a critical regulator of the adhesin of germ cell;thus,we sought to explore a novel oligoasthenospermia model based on TAp73 gene suppression.Methods:Mice in the Pifithrin-αgroup were injected intraperitoneally with 2.5 mg/kg Pifithrin-α(TAp73 inhibitor)daily for 30 consecutive days.Reproductive hormone levels and epididymal sperm quality,as well as the network morphology of Sertoli cells were tested.Results:Sperm density,motility,and the relative protein and mRNA expression of TAp73 and Nectin 2 were obviously decreased in the Pifithrin-αgroup compared with the normal control group.No significant distinction was observed in the relative mRNA and protein expression of ZO-1.Furthermore,the tight junctions(TJs)and api-cal ectoplasmic specialization(ES)were destroyed in the Pifithrin-αgroup.Conclusion:The above results indicate that we successfully established a new oli-goasthenospermia mouse model.This study provides a foundation for further explo-ration of the roles of TAp73 genes during spermatogenesis and provides new research objects for further oligospermia research and future drug discovery.
基金supported by Guangdong Science and Technology Department (No.2003B30506No.C31302)
文摘Objective To evaluate the effect of the accessory gential gland on the male fertility. Methods The relevant seminal parameters of 32 normal donors and 35 patients with olioasthenospermia were measured by seminal routine analysis, sperm morphology and accessory, gentital glands markers.Results The percentage of normal spermatozoa, the function of sperm penetration through cervical mucus and the enzyme activity of neutral α-1,4-glucosidase significantly declined as compared with the control. There was a significant negative relationship between the semen volume and the sperm motility for the oligoasthenospermia （r= -0.415, P〈0.05）, and a significant positive relationship between the sperm count and the percentage of normal sperm （r=0.393, P〈0.05）.Conclusion The abnormal function of epididymis exists in patients with oligoasthenospermia. Sperm function,for the oligoasthenospermia declines with an increase of the percentage of the abnormal sperm. The poorer the spermatogenises function of the testis is, the higher the percentage of the abnormal sperm appears.
基金Central Special Fund for Guiding Local Science and Technology Development(No.Guike ZY20198013)。
文摘Objective:To investigate the effect of Xuduan Zhongzi prescription on the expression of HSP60 and HSP90 in testicular spermatogenic cells and semen quality of oligozoospermia and asthenospermia model rats.Methods:A total of 40 SPF male rats were randomly divided into 10 blank control group and 30 model group.After successful modeling,the model group was divided into model control group,levocarnitine group and Xuduan Zhongzi prescription group with 10 rats in each group.The rats in levocarnitine group and Xuduan Zhongzi prescription group were given corresponding drugs by gavage according to the equivalent measurement of humanbody,the blank control group and model control group were given the same amount of normal saline by gavage,and the rats in each group were killed after 8 weeks of continuous administration.Take the left epididymis of each group for sperm quality detection,take the left testis of each group,observe the histomorphological changes by HE staining,and detect the expression of HSP60 and HSP90 in testicular tissue by immunohistochemistry and Western blot.Results:HE staining showed that compared with the blank control group,the convoluted tubules in the model group were significantly atrophic,degenerated and irregularly arranged,the spermatogenic epithelium showed a large number of vacuoles,Sertoli cell degeneration,obvious inflammatory infiltration in stromal cells,stromal cell proliferation,scattered rows of epithelial cells,a large number of exfoliation and degeneration,and sperm were rare.After the intervention of Xuduan Zhongzi prescription,the above lesions were significantly improved,the structure of seminiferous tubules was relatively regular and orderly,denser than that of the model group,sertoli cells and stromal cells increased significantly,the seminiferous epithelium was thicker than that of the model group,with rich layers,and a large number of sperm could be seen in the lumen.Immunohistochemistry and Western Blot showed that the expression of HSP60 and HSP90 increased significantly after the intervention of Xuduan Zhongzi prescription in model rats(P<0.01).The sperm quality test results showed that compared with the blank control group,the sperm concentration and activity rate in the model group decreased significantly,(P<0.01).After continuous seed cutting,the sperm concentration and activity rate of model rats were significantly increased,(P<0.01).Conclusion:Xuduan Zhongzi prescription may inhibit the apoptosis of testicular spermatogenic cells in oligoasthenospermia model rats by increasing the expression of HSP60 and HSP90,so as to improve sperm quality.
基金This study was funded by the Beijing University of Chinese Medicine(2018-zxfzjj-002)by the project of Beijing University of Chinese Medicine and Beijing Tong Ren Tang Company Limited Scientific Research Institute(2020071720310).
文摘Objective:To investigate the therapeutic effects and pharmacological mechanisms of Yishen Xingyang capsule(YXC)in oligoasthenospermia(OA)rats.Methods:Forty-eight male SpragueeDawley rats were randomly divided into eight groups of six rats each:normal control(NC);model control(MC);three different positive drug(PD);and low-,medium-,and high-dose YXC groups.A rat model of OA was established by administering glucosides of Tripterygium wilfordii Hook.F(GTW).After YXC administration,penile erectile function was observed.The epididymis,blood,and testes of the rats were harvested for analysis of sperm quality,sex hormone levels,mitochondrial membrane potential,and the transforming growth factor(TGF)-b1/Smad signaling pathway.Results:Compared with that in the MC group,penile erectile function in the YXC groups and three PD groups increased(all P<.01).Moreover,sperm quality in the YXC groups and three PD groups improved(all P<.001).The levels of testosterone,follicle stimulating hormone,and luteinizing hormone in the three PD and YXC groups increased(all P<.05).The mitochondrial membrane potential in the three PD and YXC groups significantly improved(all P<.001).Furthermore,the YXC and three PD groups showed decreased TGF-b1 expression(all P<.05)compared with the MC group.The high-dose YXC group and three PD groups improved Smad2 and Smad4 expression(all P<.05).Conclusion:YXC improved penile erectile function and sperm quality in OA rats,and the underlying mechanism included increase in sex hormones,inhibition of sperm apoptosis,and regulation of the TGFb1/Smad signaling pathway.Meanwhile,this study provides a new effective drug option for the treatment of OA,which is beneficial to male reproductive health and social harmony.
基金The central government guides local science and technology development projects(No.Guike ZY20198022)Guangxi University of Traditional Chinese Medicine Graduate Education Innovation Program(No.YCSZ2020013,YCSY2020051,YCXJ2021067)。
文摘Objective:To investigate the protective mechanism of Xuduan Zhongzi prescription against epididymal oxidative damage in oligoasthenospermia model rats.Methods:Forty SD rats were randomly divided into blank group,model group,Xuduan Zhongzi prescription group(10g/kg)and L-carnitine group(0.1g/kg).Except blank group,all induced oligoasmospermia.The blank group and model group were given normal saline intragastric administration,the Xuduan Zhongzi prescription group was given Xuduan Zhongzi prescription solution intragastric administration,and the L-carnitine group was given L-carnitine intragastric administration.HE staining was used to observe the epididymis structure after 8 weeks.The concentration and activity rate of epididymis sperm were measured by sperm quality.MRNA and protein expression levels of Nrf2,NQO1 andγ-GCs in epididymis were detected by RT-qPCR and immunohistochemistry.Results:①HE staining:in the blank group,the epididymis tubes were arranged tightly and regularly,the tissue structure was complete,the epithelial cells were arranged orderly,and the lumen sperm were numerous and evenly distributed.The epididymis of model group showed structural atrophy,loose arrangement,enlarged mesenchyme,increased cell debris and significantly reduced sperm cells.Compared with the model group,the lumen lesions of epididymis in Xuduan Zhongzi prescription group and L-carnitine group were significantly improved,and the amount of normal sperm in lumen was increased and the distribution was uniform.②Results of sperm quality comparison among each group:sperm density and sperm motility rate:compared with blank group,sperm density and sperm motility rate in other groups were significantly decreased(P<0.05),and sperm density and sperm motility rate in model group were significantly decreased(P<0.05);Compared with model group,the sperm density and motility rate in Xuduan Zhongzi prescription group and L-carnitine group were significantly increased(P<0.05).③RT-qPCR and immunohistochemistry:Compared with the blank group,the mRNA and protein levels of Nrf2,NQO1 andγ-GCs in epididymal rats in model group were significantly decreased(P<0.05),while the mRNA and protein levels of Nrf2,NQO1 andγ-GCs were significantly increased in L-carnitine group and Continua seed formula group(P<0.05).Conclusion:Xuduan Zhongzi prescription can reduce oxidative stress damage and improve sperm quality of oligoasthenospermia.The mechanism may related to promoting the activation of Nrf2-NQO1/γ-GCS pathway in epididymis of oligoasthenospermia rats,and up-regulate the expressions of Nrf2,NQO1 andγ-GCS proteins.
文摘目的基于AMPK介导的线粒体自噬方面探究国医大师王琦院士研发的治疗男性不育的国家中药三类新药黄精赞育胶囊治疗少弱精子症细胞层面的分子机制。方法选择丙烯醛(acrolein,ACR)作用于GC-2spd(ts)小鼠精母细胞制作少弱精子症细胞模型,通过CCK8细胞活性检测确定后续造模用ACR浓度和造模时间。造模成功后加入不同浓度含黄精赞育胶囊的完全培养基进行细胞培养,于24 h后CCK8细胞活性检测,根据细胞活力确定后续给药浓度。GC-2spd细胞贴壁后分为NC组、造模组和ACR+HJZY给药组,通过共聚焦荧光显微镜观测黄精赞育胶囊对线粒体自噬的影响;将上述3组细胞分别转染siRNA-NC和siRNA-AMPK,分为siRNA-NC+control、siRNA-NC+ACR、siRNANC+ACR+HJZY、siRNA-AMPK+control、siRNA-AMPK+ACR、siRNA-AMPK+ACR+HJZY 6组,通过Western blot验证黄精赞育胶囊对AMPK介导的p-AMPK、LC3B、P62、PINK1、Parkin、TBK1、ULK1等线粒体自噬相关蛋白的调控作用。结果通过细胞活性检测实验确定ACR的造模浓度为34μmol/L,造模时间为20 min,HJZY给药浓度为160μmol/L。共聚焦荧光显微镜显示黄精赞育胶囊对受损生精细胞线粒体膜电位具有一定正向调节作用,造模组线粒体膜电位较NC组大幅降低,ACR+HJZY给药组给药后细胞膜电位较造模组有所升高,差异有统计学意义(P<0.05);Western blot检测结果显示siRNA-NC+ACR组p-AMPK/AMPK和PINK1蛋白表达水平较siRNA-NC+control组下降(P<0.001),siRNA-NC+ACR组Parkin蛋白水平较siRNA-NC+control组有所下降,但差异无统计学意义,HJZY给药后这3种蛋白表达水平均较siRNANC+ACR组上升(P<0.001);siRNA-NC+ACR组LC3B、P62、TBK1和ULK1蛋白表达水平均较siRNA-NC+control组上升(P<0.01),siRNA-NC+ACR+HJZY组上述蛋白表达水平均较siRNA-NC+ACR组有所下降(P<0.05)。转染敲减基因siRNAAMPK后,siRNA-AMPK+ACR组p-AMPK/AMPK、PINK1和Parkin蛋白表达水平较siRNA-AMPK+control组有所下降(P<0.01);H J Z Y给药后上述3种蛋白表达水平与s i R N A-A M P K+A C R组比较差异均无统计学意义;s i R N A-AMPK+ACR+HJZY组与siRNA-AMPK+ACR组相比,LC3B蛋白表达水平仍降低(P<0.01),但P62、TBK1和ULK1蛋白表达水平差异均无统计学意义;siRNA-AMPK+control组与siRNA-NC+control组相比,p-AMPK/AMPK、ULK1和TBK1蛋白表达水平下降(P<0.001),PINK1蛋白表达水平亦下降(P<0.05),P62蛋白表达水平上升(P<0.001);siRNA-AMPK+ACR组与siRNA-NC+ACR组相比,TBK1蛋白表达水平下降(P<0.001),LC3B蛋白表达水平下降(P<0.01),ULK1蛋白表达水平亦下降(P<0.05),PINK1和Parkin蛋白表达水平有所下降但差异无统计学意义;siRNA-AMPK+ACR+HJZY组与siRNANC+ACR+HJZY组相比,p-AMPK/AMPK、PINK1和Parkin蛋白表达水平下降(P<0.05),LC3B蛋白表达水平亦下降(P<0.01),P62蛋白表达水平上升(P<0.001),TBK1和ULK1蛋白表达水平差异无统计学意义。结论黄精赞育胶囊可能通过调节AMPK介导的线粒体自噬达到治疗男性少弱精子症的效果。
基金co-supported by National Outstanding Youth Science Fund Project of National Natural Science Foundation of China(No.82022073)National Natural Science Foundation of China(No.82174389)+1 种基金Natural Science Foundation of Beijing Municipality(No.7202115,China)Postdoctoral Research Foundation of China(No.2021M690474)。
文摘Oligoasthenospermia is the primary cause of infertility.However,there are still enormous challenges in the screening of critical candidates and targets of oligoasthenospermia owing to its complex mechanism.In this study,stem cell factor(SCF),c-kit,and transient receptor potential vanilloid 1(TRPV1)biosensors were successfully established and applied to studying apoptosis and autophagy mechanisms.Interestingly,the detection limit reached 2.787×10^(-15)g/L,and the quantitative limit reached 1.0×10^(-13)g/L.Furthermore,biosensors were used to investigate the interplay between autophagy and apoptosis.Schisandrin A is an excellent candidate to form a system with c-kit similar to SCF/c-kit with a detection constant(K_(D))of 5.701×10^(-11)mol/L,whereas it had no affinity for SCF.In addition,it also inhibited autophagy in oligoasthenospermia through antagonizing TRPV1 with a K_(D) of up to 4.181×10^(-10)mol/L.In addition,in vivo and in vitro experiments were highly consistent with the biosensor.In summary,high-potency schisandrin A and two potential targets were identified,through which schisandrin A could reverse the apoptosis caused by excessive autophagy during oligoasthenospermia.Our study provides promising insights into the discovery of effective compounds and potential targets via a well-established in vitro-in vivo strategy.
